Dynein-adaptor interaction mechanisms and malfunction at atomic resolution

项目摘要 神经退行性疾病大多是无法治愈的疾病，会导致进行性变性或 神经细胞死亡。脊髓性肌萎缩症(SMA)等突出例子是由 细胞质动力蛋白多蛋白复合体的突变。动力蛋白1参与了广泛的 细胞活动，从蛋白质、RNA和囊泡的货物运输到核迁移 和细胞分裂。动力蛋白沿微管的行进运动需要结合到 基本的共激活动力蛋白和特定的接头蛋白，它们招募货物并促进运动 通过与动力蛋白和动力肌动蛋白形成稳定的三元络合物。我们对这些互动的理解是 仍然存在许多有限的问题：货物适配器如何将动力蛋白和动力蛋白结合在一起？多么 这是否会导致马达的激活和运输的启动？哪些因素决定了 适配器专一性？致病突变是如何改变这些相互作用的？重要的是，它是 越来越明显的是，与动力蛋白1的轻质中间链(LIC1)结合是一种常见的 功能截然不同的适配器的特点。我们将重点调查两国之间的相互作用 LIC1的C-端尾，带有细长的适配器，BICD2和Hook3，它们各自具有重要的但 在运输中具有独特的功能。这些研究之所以复杂化，是因为C-C的无序性质 终端LIC1使从X射线获得原子分辨率的全面动态图像变得困难 或EM技术。然而，我们将采取创新的方法，使用核磁共振 (核磁共振)是一个纳米尺度的‘放大镜’，可以精确定位蛋白质内部的原子相互作用部位。 决议。与其他方法相比，核磁共振特别适合于研究无序蛋白质和 描述高度动态的交互作用。我们提出了四个目标：(1)结构和动力 游离C-末端动力蛋白LIC1的表征。(2)动力蛋白LIC1与LIC1相互作用的特征 Spindly、BicD2和Hook3以及它们的结合竞争。(3)效果的预测和检验 动力蛋白LIC1-接头相互作用的突变。(4)相互作用三角的结构和动力学基础 在动力蛋白LIC1、纤毛状和Rod/Zw10/ZWilch复合体之间。因此，我们的实验是可以期待的 为人类动力蛋白LIC1如何与结构多样化的结合提供详细的分子洞察力 动力蛋白适配器。通过合作，我们将把我们的发现扩展到动物模型中。总体而言，这 这项研究意义重大，因为动力蛋白和适配器中存在致病点突变 蛋白质表明，这些洞察力也将有助于理解由功能障碍引起的疾病 动力蛋白驱动的货物运输，并将提供治疗干预目标。

Project Summary Neurodegenerative diseases are mostly incurable conditions that result in progressive degeneration or death of nerve cells. Prominent examples such as Spinal Muscular Atrophy (SMA) are caused by mutations in the cytoplasmic dynein multi-protein complex. Dynein 1 participates in a wide array of cellular activities, ranging from the cargo transport of proteins, RNA, and vesicles to nuclear migration and cell division. Processive movement of dynein along microtubules requires the binding to the essential co-activator dynactin and specific adaptor proteins, which recruit cargo and facilitate movement by forming a stable ternary complex with dynein and dynactin. Our understanding of these interactions is limited and many questions remain: How do cargo adaptors bring dynein and dynactin together? How does this lead to activation of the motor and the initiation of transport? Which elements determine the adaptor specificity? How do the disease-causing mutations modify these interactions? Importantly, it is becoming increasingly evident that binding to light intermediate chain of dynein 1 (`LIC1') is a common feature of functionally distinct adaptors. We will focus our investigations on the interaction between the C-terminal tail of the LIC1 with the adaptors Spindly, BICD2 and Hook3, which each have important but distinct functions in transport. These studies are complicated by the fact that the disordered nature of C- terminal LIC1 makes it difficult to obtain a comprehensive dynamic view at atomic resolution from X-ray or EM techniques. However, we will take the innovative approach of using nuclear magnetic resonance (NMR) as a nano-scale `magnifying glass' that pinpoints interaction sites within the proteins at atomic resolution. Compared to other methods, NMR is uniquely suited to study disordered proteins and to characterize highly dynamic interactions. We propose four aims: (1) Structural and dynamical characterization of free C-terminal dynein LIC1. (2) Characterization of dynein LIC1 interaction with Spindly, BicD2 and Hook3, and their binding competition. (3) Prediction and testing of effects of mutations on dynein LIC1 - adaptor interaction. (4) Structural & dynamic basis of interaction triangle between dynein LIC1, Spindly and ROD/Zw10/Zwilch complex. Our experiments are therefore expected to provide detailed molecular insight into how human dynein LIC1 engages with structurally diverse dynein adaptors. Through collaboration, we will extend our findings into animal models. Overall, this research is significant because the presence of pathogenic point mutations in dynein and adaptor proteins suggests that these insights will also help to understand the diseases caused by malfunction of the dynein-driven cargo transport and will offer therapeutic intervention targets.